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@article{223837,
author = {Rotrekl, Vladimír and Nejedlá, Eliška and Kučera, Igor and Abdallah, Fuad and Palme, Klaus and Brzobohatý, Břetislav},
article_number = {3},
keywords = {beta-glucosidase; cysteine residues; disulfide bridge; structure-function relationships},
language = {eng},
issn = {0014-2956},
journal = {European Journal of Biochemistry},
title = {The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase},
volume = {266},
year = {1999}
}
TY - JOUR
ID - 223837
AU - Rotrekl, Vladimír - Nejedlá, Eliška - Kučera, Igor - Abdallah, Fuad - Palme, Klaus - Brzobohatý, Břetislav
PY - 1999
TI - The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase
JF - European Journal of Biochemistry
VL - 266
IS - 3
SP - 1056
EP - 1056
SN - 00142956
KW - beta-glucosidase
KW - cysteine residues
KW - disulfide bridge
KW - structure-function relationships
N2 - The maize Zm-p60.1 gene encodes a beta-glucosidase that can release active cytokinins from their storage forms, cytokinin-O-glucosides. Mature catalytically active Zm-p60.1 is a homodimer containing five cysteine residues per a subunit. Their role was studied by mutating them to alanine (A), serine (S), arginine (R), or aspartic acid (D) using site-directed mutagenesis and subsequent heterologous expression in Escherichia coli. All substitutions of C205 and C211 resulted in decreased formation and/or stability of the homodimer, manifested as accumulation of high levels of monomer in the bacterial expression system. Examination of urea- and glutathione-induced dissociation patterns of the homodimer to the monomers, HPLC profiles of hydrolytic fragments of reduced and oxidized forms, and a homology-based three-dimensional structural model revealed that an intramolecular disulfide bridge formed between C205 and C211 within the subunits stabilized the quaternary structure of the enzyme. Mutating C52 to R produced a monomeric enzyme protein, too. No detectable effects on homodimer formation were apparent in C170 and C479 mutants. Given the Km values for C170A/S mutants were equal to that for the wild-type enzyme, C170 cannot participate in enzyme-substrate interactions. Possible indirect effects of C170A/S mutations on catalytic activity of the enzyme were inferred from slight decreases in the apparent catalytic activity, k'cat. C170 is located on a hydrophobic side of an alpha-helix packed against hydrophobic amino acid resides of beta-strand 4, indicating participation of C170 in stabilization of a (beta/alpha)8 barrel structure in the enzyme. In C479A/D/R/S mutants, Km and k'cat were influenced more significantly suggesting a role for C479 in enzyme catalytic action.
ER -
ROTREKL, Vladimír, Eliška NEJEDLÁ, Igor KUČERA, Fuad ABDALLAH, Klaus PALME and Břetislav BRZOBOHATÝ. The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase. \textit{European Journal of Biochemistry}. 1999, vol.~266, No~3, p.~1056-1065. ISSN~0014-2956.
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